A previously identified sterol catabolic gene cluster is widely dispersed among actinobacteria, enabling them to degrade and grow on naturally occurring sterols. We investigated the physiological roles of various genes by targeted inactivation in mutant RG32 of Rhodococcus rhodochrous, which selectively degrades sterol side chains. The ltp3 and ltp4 deletion mutants were each completely blocked in side chain degradation of β-sitosterol and campesterol, but not of cholesterol. These results indicated a role of ltp3 and ltp4 in the removal of C24 branches specifically. Bioinformatic analysis of the encoded Ltp3 and Ltp4 proteins revealed relatively high similarity to thiolase enzymes, typically involved in β-oxidation, but the catalytic residues characteristic for thiolase enzymes are not conserved in their amino acid sequences. Removal of the C24-branched side chain carbons of β-sitosterol was previously shown to proceed via aldolytic cleavage rather than by β-oxidation. Our results therefore suggest that ltp3 and ltp4 likely encode aldol-lyases, rather than thiolases. This is the first report on the molecular characterization of genes with specific and essential roles in carbon-carbon bond cleavage of C24-branched chain sterols in Rhodococcus strains, most likely acting as aldol-lyases. The results provide a clear contribution to our understanding of sterol degradation in actinobacteria.